Polypeptide compound which binds to glyco-conjugates and to artificial pertussis toxin antigen

ABSTRACT

A polypeptide of the formula 
     
         H--X.sup.1 
    
      --Gln--Thr--Art--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X 2  --Y 
     in which X 1  and X 2  each represents an optional coupling-facilitating amino acid residue, and Y represents --OH or --NH 2 , and an artificial compound in free or carrier-associated form with the capability of binding to glyco-conjugates, especially immunoglobulins, which compound is chosen from the group consisting of said peptide and functional analogues and functional derivatives thereof, are disclosed. Additionally, there is described an artificial pertussis toxin antigen, which mainly consists of at least one peptide sequence reacting with antibodies induced by the native pertussis toxin selected from the above polypeptide and parts thereof. The above described artificial pertussis toxin antigen is included as a diagnostic antigen in a diagnostic immunoassay kit for the determination of antibodies induced by the native pertussis toxin in a sample of biological fluid, and as an immunizing component in a vaccine composition against whooping cough, respectively. Furthermore, there is described an intradermal skin test composition comprising the above described artificial pertussis toxin antigen.

The present invention relates to a new polypeptide, to an artificialcompound, selected from the new peptide and functional analogues andderivatives of the peptide, in free or carrier-associated form with thecapability of bindning to glyco-conjugates, especially immunoglobulins,to artificial pertussis toxin antigens, which mainly consist of peptidesequences reacting with antibodies induced by the native pertussis toxinselected from the new polypeptide and parts thereof, to a diagnosticimmunoassay kit comprising as a diagnostic antigen, said antigensreacting with antibodies induced by the native pertussis toxin, to avaccine composition comprising as an immunizing component antigensselected from said antigens reacting with antibodies induced by thenative pertussis toxin, and to an intradermal skin test compositioncomprising antigens selected from said antigens reacting with antibodiesinduced by the native pertussis toxin.

BACKGROUND

In the field of immunology it is well known that most biologicalorganisms produce specific proteins which selectively and specificallyrecognize various protein and/or carbohydrate structures. Examples ofsuch specific proteins derived from bacteria are Protein A and Protein Gwhich both bind to certain immunoglobulins from various species.Examples of such specific proteins derived from plants or lowerinvertebrates are so-called lectins which bind to carbohydratestructures of immunoglobulins and other glyco-conjugates. Examples ofsuch lectins are Concanavalin A, Wheat germ agglutinin,Phytohaemagglutinin and Helix pomatia lectin.

Each of the above mentioned specific proteins bind to a specific groupof protein structures and/or carbohydrate structures.

The present invention provides an artificial compound which has thecapability of binding to glyco-conjugates, especially immunoglobulins.

Up to now no peptide antigens constituting part of pertussis toxin havebeen identified in the art. Since such antigens have not been provided,it has not been possible to develop diagnostic immunoassay kitscomprising such antigens as diagnostic antigens nor to develop vaccinesagainst whooping cough based on such antigens.

Diagnosis of whooping cough with the aid of antigens directed againstBordetella pertussis antibodies or proteins produced by B. pertussishave been published, but as diagnostic antigen there has been usedfimbrial hemagglutinin (see e.g. Granstrom, M., Granstrom, G., Lindfors,A, and Askelof, P. 1982. Serologic diagnosis of whooping cough by anenzyme-linked immunosorbent assay using fimbrial hemagglutinin asantigen. J. Infect. Dis. vol 146: 741-745), or sonicated B. pertussisbacteria (see e.g. Goodman, Y. E., Wort, A. J. and Jackson, F. L. 1981.Enzyme-linked immunosorbent assay for detection of pertussisimmunoglobulin A in nasopharyngeal secretions as an indicator of recentinfection J. Clin. Microbiol. vol. 13: 286-292, and Viljanen, M. K.,Ruuskanen, O., Granberg, C. and Salmi, T. T. 1982. Serological diagnosisof pertussis: IgM, IgA and IgG antibodies against Bordetella pertussismeasured by enzyme-linked immunosorbent assay. Scand. J. Infect. Dis.vol. 14: 112-117).

As is well known in the art currently used vaccines against whoopingcough are in USA and many other countries based on inactivatedBordetella pertussis bacteria. M. Pittman proposed 1979 that whoopingcough was mediated by an exotoxin (pertussis toxin) (see Pittman, M.1979. Pertussis toxin: The cause of the harmful effects and prolongedimmunity of whooping cough. A hypothesis. Rev. Infect. Dis. vol.1:401-412) and in Japan acellular vaccines comprising inactivatedpertussis toxin are currently in use.

Recently the nucleotide sequence of pertussis toxin was published(Locht, C. and Keith, J. M., 1986. Pertussis Toxin Gene: NucleotideSequence and Genetic Organization, Science, vol. 232, p. 1258-1264). Inthis article the authors suggest i.a. that synthetic oligopeptides thatinclude protective epitopes also will be useful in the development of anew generation of vaccines, but there is no teaching or suggestion ofsuch epitopes.

Another recently published article concerning pertussis toxin genes is:Nicosia, A., Perugini, M., Franzini, C., Casagli, M. C., Borri, M. G.,Antoni, G., Almoni, M., Neri, P., Ratti, G., and Rappuoli, R., 1986.Cloning and sequencing of the pertussis toxin genes: Operon structureand gene duplication. Proc. Natl. Acad. Sci. USA, vol. 83, 4631-4635. Inthis publication it is stated i.a. that "Manipulation of the toxin geneby genetic engineering could be a way to produce large amounts ofdetoxified protein". This is merely a suggestion and no manipulatedtoxin gene is disclosed.

Yet another publication in this field is: Engstrom, O., Rodmalm, K.,Jornvall, H., Lundquist, G., Kalman, M., Simonscits, A., Bartfai, T.,Lofdahl, S., and Askelof, P., 1986. Characterization of the N-terminalstructure of pertussis toxin subunit S1 and hybridization ofoligodeoxyribonucleotide probes with Bordetella pertussis DNA fragment,FEMS Microbiology Letters, vol. 36, 219-223. Also this article makessuggestions, namely, "The gene may also be introduced into otherorganisms for production of toxin. Sequencing of the gene would allowsynthesis of peptides corresponding to the antigenic epitopes of thetoxin and hence to the development of a synthetic pertussis vaccine."However, the antigenic epitopes of the pertussis toxin have not beenidentified, synthesized nor tested.

As regards intradermal skin test compositions, such compositions fortesting immunity against pertussis are hitherto not described in theart.

DESCRIPTION OF THE INVENTION

In one aspect of the invention there is provided a new polypeptide ofthe formula ##STR1## in which X¹ and X² each represents an optionalcoupling--facilitating amino acid residue, and Y represents --OH or--NH₂.

Examples of suitable optional amino acid residues are --Lys-- and--Cys--. These optional amino acid residues facilitate the coupling of acarrier, such as bovine serum albumin, to said polypeptide.

The peptide according to the invention has been synthesized inaccordance with per se known solid phase techniques.

As is well known in this field a peptide is synthesized either in acid(COOH) or amide (CONH₂) form depending on available resin-bound startingamino acid.

In another aspect of the invention there is provided an artificialcompound in free or carrier-associated form with the capability ofbinding to glyco-conjugates, which compound is chosen from the groupconsisting of the polypeptide of the formula

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represents an optional coupling--facilitatingamino acid residue, and Y represents --OH or --NH₂ ; and functionalanalogues and functional derivatives thereof.

When the compound according to the invention is in carrier-associatedform it can be associated to any carrier to which it can be linked byphysical/chemical interaction, such as covalent binding, ionic binding,hydrogen binding or hydrophobic binding. Examples of such carriers aremineral carriers, e.g. glass, aluminium hydroxide, calcium phosphate,etc., plastic surfaces, e.g. microplates, beads, etc., lipids,liposomes, carbohydrates, amino acids, peptides, proteins, membranes orfractions thereof and whole cells or fractions thereof.

The expression "glyco-conjugate" as it is used in this specification andclaims is intended to include glycoproteins, glycolipids,peptidoglycanes and proteoglycanes whether they are in free orcarrier-associated form (both in vivo and in vitro).

The binding mechanism is not yet ascertained, but it is firmly believedthat the compound according to the invention binds to the glyco portionof the above defined glyco-conjugates, even though there may also occureother sites on the glyco-conjugates which may be of equal importance inthe interaction between said compound and the glyco-conjugates. It isfurther believed that it is the binding conformation of the peptide ofthe invention in aqueous solution (pH-values ranging from 1 to 13) thatis responsible for this specific binding to the glyco-conjugates.

In view of the above the compound of the invention can be selected froma wide variety of compounds as long as they have a similar capability ofbinding to glyco-conjugates (e.g. glycoproteins) as the peptide of theinvention.

Thus the expression "functional analogues" of the peptide of theinvention is intended to cover i.a. shorter or longer monomers orpolymers of the peptide (e.g. functional fractions or fragments, orpolymers of fractions or fragments of the peptide) with or withoutsubstitution of one or several amino acid residues for other amino acidresidues, as long as the analogues have a similar capability of bindingto glyco-conjugates (especially glycoproteins e.g. immunoglobulins) asthe peptide of the invention.

Further the expression "functional derivatives" of the peptide of theinvention is intended to cover compounds having a similar capability ofbinding to glyco-conjugates (especially glycoproteins e.g.immunoglobulins) as the peptide of the invention. Examples of suchcompounds are compounds having essentially the structure of the peptideaccording to the invention, but having one or several amino acidresidues substituted for other chemical groups, i.e. organic as well asinorganic molecules or elements.

Since it is believed that it is the binding conformation of the peptideof the invention in aqueous solution that is responsible for the bindingof said peptide to the defined glyco-conjugates, it follows that thefunctional analogues and functional derivatives (of the peptide of theinvention) should comprise at least one conformation correspondingessentially to the binding conformation of said peptide in aqueoussolution.

In a preferred embodiment of the invention there is provided anartificial compound in free or carrier--associated form with thecapability of binding to immunoglobulins, which compound is chosen fromthe group consisting of the polypeptide of the formula

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represents an optional coupling--facilitatingamino acid residue, and Y represents --OH or --NH₂ ; and functionalanalogues and functional derivatives thereof.

The polypeptide and the compound according to the invention have highlyinteresting properties which make them extremely useful in the field ofimmunology.

The compound of the invention binds to glyco--conjugates, especiallyglycoproteins e.g. immunoglobulins, and can thus be used inenzyme-associated form in enzyme-linked immunosorbent assay (ELISA)instead of conjugates conventionally used in such assays.

The compound of the invention can be used as a general sorbent forglyco-conjugates, e.g. immunoglobulins or other immunologically activesubstances, e.g. β2-microglobulin. As a sorbent it can be applied to orutilized for extracorporeal body fluids (in vivo) using plasmapheresisto clear the body fluid from immune complexes. Other examples of theusefulness of said compound as a sorbent in plasmapheresis is in thetreatment of such diseases as allergies, leukemias and AIDS.

As a sorbent it can further be used (in vitro) in purifying poly- andmonoclonal antibodies and other immunologically active substances.

Further the compound of the invention in carrier--associated form has awide applicability (in vitro) in the field of immunochemistry. Thecarrier can specifically be a label (marker), such as gold particles,colloidal gold, enzymes (e.g. alkaline phosphatase or horseradishperoxidase), avidin, biotin or some radioactive isotope, (e.g. iodine).

The compound of the invention binds to immunologically active cells andhence it can be used in carrier-associated form for targeting (in vivo).Examples of carriers in this respect are various pharmaceuticals,cytostatic agents and hormones. An application of targeting of specialinterest is targeting of artificial, synthetic and subunit vaccines toimmune-competent cells.

The compound according to the invention can further be used to blockreceptors on cell membranes, a property which makes said compound usefulfor immunosuppression in patients receiving allografts, such as kidney,liver or bone-marrow.

A further interesting property of the compound of the invention in freeform is that it behaves as a lymphocyte activating factor (LAF) andinduces mitosis in cells and thus the compound possesses a strongadjuvant activity.

In another aspect of the invention there is provided an artificialpertussis toxin antigen, which mainly consists of at least one peptidesequence reacting with antibodies induced by the native pertussis toxinselected from the group consisting of the polypeptide

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represents an optional coupling--facilitatingamino acid residue, and Y represents --OH or --NH₂ ; and parts thereof.

The polypeptide according to the invention is able to induce antibodies,which react with the native toxin, in animals. Hence it can beconsidered as an antigen. Being an antigen, the polypeptide according tothe invention is likely to include shorter peptide sequences which ontheir own induce antibodies which react with the native pertussis toxin.The artificial pertussis toxin antigen reacting with antibodies inducedby the native pertussis toxin according to the invention does notnecessarily comprise more than one such shorter peptide sequence beingpart of the polypeptide of the invention together with a carrier, eventhough it preferably comprises several such shorter peptide sequences.

The expression "artificial pertussis toxin antigen", as it is used inthis specification and appended claims, is contemplated to includepertussis toxin antigens that have been produced in an artificialmanner, i.e. contrived through human effort and not by natural causesdetached from human agency. Even though the peptide sequencesconstituting, or constituting part of, the artificial pertussis toxinantigen according to the invention have been chemically synthesizedaccording to per se known solid-phase technique, said peptide sequencescan be produced using some other techniques, e.g. synthesis in liquidphase by coupling one amino acid to the next in known manner,degradation, cloning etc, and it is intended that the expression"artificial" should cover products produced by any such technique.

The word "comprises" is used, in this specification and appended claims,to indicate that something is included, but that that something does notnecessarily constitute the only thing included.

The expression "mainly consists of" in conjunction with "peptidesequences reacting with antibodies induced by the native pertussistoxin" is used to indicate that the ability of the artificial pertussistoxin antigen to react with antibodies induced by the native pertussistoxin derives from said peptide sequences".

The word "carrier" should be interpreted broadly, and the carrier can beanything to which the peptide in question can be linked byphysical/chemical interaction, such as covalent binding, ionic binding,hydrogen binding or hydrophobic binding. Examples of such carriers aremineral carriers, e.g. aluminium hydroxide, calcium phosphate, etc.,plastic surfaces, e.g. microplates, beads, etc., lipids, liposomes,carbohydrates, amino acids, peptides and proteins.

In still another aspect of the invention there is provided a diagnosticimmunoassay kit for the determination of antibodies induced by thenative pertussis toxin in a sample of biological fluid. The kitcomprises as a diagnostic antigen at least one antigen selected from theartificial antigens reacting with antibodies induced by the nativepertussis toxin according to the invention. Depending on the immunoassayused for the determination of antibodies induced by the native pertussistoxin the kit may comprise other suitable reagents, such as a carrier towhich said diagnostic antigen is coupled, a positive standard serumsample, a negative standard serum sample, an enzyme conjugate, such asalkaline phosphatase or peroxidase, substrate for the enzyme conjugate,such as paranitrophenylphosphate, agar or agarose gel, radioactivlylabelled antigen, buffer solutions and/or washing solutions. Optionallyall the reagents in the kit are contained in separate sealed test tubesor vials marked with specific labels.

The sample of biological fluid is preferably a nasopharyngeal secretion,saliva, blood or serum sample from an animal, e.g. a human.

Examples of immunoassays in which the kit according to the invention canbe used are ELISA (enzyme-linked immunosorbent assay), Immunodiffusion,Radioimmunoassay (RIA), and Immunoelectrophoresis (IE).

When ELISA (enzyme-linked immunosorbent assay) is used the kit accordingto the invention will comprise

a) a diagnostic antigen of the invention

b) optionally a carrier for said diagnostic antigen

c) optionally a positive standard serum sample

d) optionally a negative standard serum sample

e) an enzyme conjugate

f) optionally a substrate for said enzyme conjugate

g) optionally buffer solution(s), and

h) optionally washing solution(s).

When immunodiffusion or immunoelectrophoresis (IE) is used the kitaccording to the invention will comprise the same as for ELISA, with theexception of items e) and f). Instead there is needed a gel, such asagar or agarose gel, but such a gel is normally not included in the kit,since it is commonly available.

When radioimmunoassay (RIA) is used the kit according to the inventionwill comprise the same as for ELISA, with the exception of items e) andf), which will be substituted for radioactively labelled antigen.Optionally there may also be included a solution for the precipitationof radioactively labelled antigen bound to antibodies, such astrichloroacetic acid or secondary antibodies.

In a further aspect of the invention there is provided a vaccinecomposition, which as an immunizing component comprises at least oneantigen selected from the artificial pertussis toxin antigens reactingwith antibodies induced by the native pertussis toxin, according to theinvention, preferably in an amount effective to protect a subject fromthe disease whooping cough, and a nontoxic pharmaceutically acceptablecarrier and/or diluent. The carrier is a carrier which has been definedherein above, and the diluent may be a conventional diluent used in theart, such as saline solution. The vaccine composition accordning to theinvention may further comprise an antigen adjuvant in an amount whichtogether with the amount of said antigen is effective to protect asubject from the disease whooping cough. Examples of commonly usedadjuvants in vaccines for humans are so-called mineral carriers, e.g.phosphate or hydroxide of calcium or aluminium, to which the antigen inquestion is adsorbed. An example of commonly used veterinary vaccineadjuvant is Freund's Complete Adjuvant. The vaccine composition mayadditionally comprise buffer(s) and/or preservative(s) as appropriate,and suitable buffers and preservatives are disclosed in e.g. USPharmacopoeia.

In still a further aspect of the invention there is provided anintradermal skin test composition, which comprises at least one antigenselected from the artificial pertussis toxin antigens reacting withantibodies induced by the native pertussis toxin, according to theinvention, in an amount effective to produce an immunological skinreaction at a specific antibody titre in a subject, and a nontoxicpharmaceutically acceptable carrier and/or diluent. The skin testcomposition according to the invention may further comprise buffer(s)and/or preservative(s), as appropriate.

Useful carriers, diluents, buffers and preservatives are defined hereinabove.

SYNTHESIS OF THE PEPTIDE ACCORDING TO THE INVENTION

Solid-phase (Merrifield) synthesis of the peptide according to theinvention has been performed in conventional manner, by coupling theamino acids to one another, whereby peptide bonds are formed, startingwith a solid phase (resin) to which the C-terminal of the first aminoacid is coupled, whereupon the C-terminal of the next amino acid iscoupled to the N-terminal of the first amino acid, etc. Finally, thebuilt-up peptide is released from the solid phase.

Specifically the following peptide was synthesized:

    H--Lys--Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--OH

Synthesis of the peptide fragments was achieved using a Biosearch SamTwo (Biosearch, Inc. California USA) peptide synthesis machine usingversion 2.38 of Biosearch's peptide synthesis automation software fort-BOC amino acids. The standard (default) coupling constants were usedfor one gram of starting material (degree of substitution=0.33 meq/g).

The resin used for the synthesis of the peptide was a standardMerrifield Resin. The suitably protected amino acids were:

    ______________________________________                                        Boc--Ala--OH     Boc--Arg(Tos)--OH                                            (Boc--L-Alanine) (a-Boc--N-Tosyl-L-Arginine)                                  Boc--Asn--OH     Boc--Gln--OH                                                 (Boc--L-Asparagine)                                                                            (Boc--L-Glutamine)                                           Boc--Lys(CL--Z)--OH                                                                            Boc--Pro--OH                                                 (Boc-e-o-Chloro-Benzyl-                                                                        (Boc--L-Proline)                                             oxycarbonyl-L-Lysine)                                                         Boc--Ser(Bzl)--OH                                                                              Boc--Thr(Bzl)--OH                                            (Boc--O-Benzyl-L-Serine)                                                                       (Boc--O-Benzyl-L-Threonine)                                  Boc--Tyr(Cl.sub.2 --Bzl)--OH                                                                   Boc--Val--OH                                                 (Boc--O-2,6-Dichloro-                                                                          (Boc--L-Valine)                                              benzyl-L-Tyrosine)                                                            ______________________________________                                    

As to the details regarding amino acid concentration, solvent volumes,reaction times etc, the recommendations of the manufacturer (Biosearch)were followed.

Specifically the following chemicals listed below were use in thesynthesis, cleavage and purification of the synthetic peptide.

    ______________________________________                                        SUBSTANCE      GRADE         SUPPLIER                                         ______________________________________                                        Acetyl imidazole                                                                             p.a.          Aldrich                                          Dimethyl formamide                                                                           p.a.          Fluka                                            Diisopropylethyl amine                                                                       Double-distilled                                                                            Aldrich                                                         from 1 g/l nin-                                                               hydrin, then                                                                  5 g/l KOH. First                                                              10% destillate                                                                discarded.                                                     Hydroxybenzyl triazole                                                                       p.a.          Aldrich                                          Methylene chloride                                                                           HPLC          Fisons (UK)                                      Ninhydrin      p.a.          Aldrich                                          Potassium hydroxide                                                                          p.a.                                                           t-Boc amino acids            Bachem                                           Hydrogen fluoride            AGA                                              Resorcinol                                                                    Dimethyl sulfide                                                              Hydroxybenzyl triazole                                                                       p.a. (synthesis)                                                                            Aldrich                                          Trifluoroacetic acid                                                                         sequanal (HPLC)                                                                             Aldrich                                          Acetonitrile   HPLC          Rathburn                                         Acetic acid    p.a.          Merck                                            Diethyl ether                Fluka                                            ______________________________________                                    

The completed resin-bound peptide was removed from the synthesis chamberand rinsed with methanol, and dried to constant weight under vacuum.

Cleavage of the Peptide from the Resin Support

Portions (0.5 g) of the washed and dried resin--bound peptide wastransferred to a cylindrical polytetrafluoroethylene (PTFE) chambercontaining 1 g resorcinol, 6.5 ml dimethyl sulfide and a PTFE- coatedmagnet. The chamber was cooled to -80° C. in a dry ice-ethanol bath.Hydrofluoric acid (HF) (final volume 10 ml) was then admitted to thechamber, which was then kept at 0° C. for 120 min on a magnetic stirrer.The acid was then removed using a water aspirator. The chamber was onceagain cooled to -80° C. and 10 ml HF were admitted to the chamber, whichwas placed over a magnetic stirrer at 0° C. for 45 min. The acid wasagain removed using a water aspirator, and the remaining mixturetransferred to a sintered glass filter. The resin/peptide mixture wasthen washed with 100 ml 10% (v/v) acetic acid and then with 50 mldiethyl eter. The acetic acid wash was also washed with 50 ml diethylether in a separatory funnel, and the ether phase removed from theaqueous phase. The pooled ether washes and the acetic acid wash werethen evaporated and lyophilised, respectively, and the peptide productrecovered. The peptide was recovered from the aqueous phase only.

The crude product was stored in glass vials in a desiccator at roomtemperature.

Purification of the Synthetic Peptide

The various components of the crude synthesis product were resolved byapplying them to a reverse-phase C-18 silica high-pressurechromatography column using acetonitrile/H₂ O (0.1% v/v Trifluoroaceticacid (TFA)) as the mobile phase. 20 μg- 100 mg of the crude peptide weredissolved in up to 1 ml H₂ O (0.1% TFA), centrifuged and injected into a1 ml loop on a Rheodyne (California, USA) HPLC injector. The solutionwas pumped through a custom-packed 10×500 mm Oligosil reverse-phase C-18column (Skandinaviska Genetec, Sweden) using a 2152 gradient controllerand two 2150 HPLC pumps (LKB, Sweden). The gradient was linear from0-100% acetontrile in 60 min. The flow rate was 2 ml/min. The elution ofthe synthesis product was monitored at 206-238 nm using an LKB 2131variable wavelength absorption monitor. Fractions were collected by handand repurified if necessary by lyophilisation of the relevant fractions,redissolution and repeating of the chromatographic procedure. The finalproduct was lyophilised and stored in glass vials at -20° C.

PREPARATION OF PEPTIDE ANTIGEN FOR IMMUNOASSAY

The above synthesized peptide was coupled to a carrier, i.e. bovineserum albumin (BSA) in the following manner to form a peptide antigen(coating antigen) which was used in ELISA.

One mg of peptide and 3.6 mg of BSA were dissolved in 2.0 ml ofphosphate buffered saline (PBS), pH 7.4.

To this solution, 30 microliters of a 2.5% (w/v) aqueousglutardialdehyde solution was added.

The reaction mixture was incubated at room temperature (20°-25° C.) forone hour, stopped by the addition of 0.5 ml of a 5 M aqueousethanolamine solution and then dialyzed against one liter of PBS at +4°C. for four hours, with changes of the dialysing fluid after 30 minutesand after two hours.

Next the content of the dialysis bag was gelfiltrated through a column(2.5×60 cm) containing Sephacryl.sup.® S-300 gel (Pharmacia, Uppsala,Sweden) equilibrated with PBS. Fractions of 3.0 ml were collected.

Fractions containing coupled material were pooled and the pooledmaterial was used in ELISA as coating antigen and was added directly,without any previous dilution, to the microplate.

ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA:s)

Bellow follows a general description of the ELISA:s. The conjugate andthe dilution of the conjugate, and the time elapsed before themicroplates were read may vary between the various assays. Thedifferences are given after the general description.

GENERAL DESCRIPTION OF ELISA EQUIPMENT

Microtitreplates (Dynatech, mod. nr 129B).

Antigen for coating of microplates (e.g. peptides and proteins).

Coatingbuffer: Phosphate buffered saline (PBS). Incubation buffer:PBS+0.05% Tween 20 (v/v) Aqueous washing fluid: 0.9% NaCl+0.05% Tween 20Serum (e.g. human and animal reference and test sera) Conjugate (e.g.alkaline phosphatase conjugated swine anti-human IgG antibodies (OrionDiagnostica) and goat anti-rabbit Ig antibodies (Sigma).

Enzyme substrate: p-nitrophenylphosphate tablets (Sigma), 1 mg/ml ofsubstrate buffer (c.f. below) Substrate buffer: 1 M aqueousdiethanolamine, pH 9.8,+0.5 mM MgCl₂ +0.02% NaN₃. Pipettes and testtubes

PERFORMANCE 1. Coating of microplates. Microplates were incubated with0.1 ml of antigen in PBS at room temperature (20°-25° C.) over night.

2. Incubation with bovine serum albumin (BSA) and serum. The plates werewashed four times and then incubated with 0.1 ml of 1% (w/v) BSA in PBSat 37° C. for one hour. After washing, 0.1 ml of serum appropriatelydiluted in incubation buffer, was added to the wells and the plate(s)was incubated at room temperature for one hour.

3. Incubation with conjugate. After washing, 0.1 ml of conjugateappropiately diluted in incubation buffer, was added to the wells andthe plate(s) was incubated at room temperature for two hours.

4. After washing, 0.1 ml of the enzyme substrate solution was added tothe wells. The plates were kept at room temperature and the absorbanceat 405 nm was read after the time indicated below.

SPECIFIC DESCRIPTIONS OF ELISA ENZYME-LINKED IMMUNOSORBENT ASSAYS

Enzyme-linked immunosorbent assay (ELISA) for measuring the reactionbetween the synthetic peptide and antibodies in rabbit or human sera, orthe reaction between the synthetic peptid and conjugate only.

The peptide used as coating antigen in the ELISA was first treated asdescribed under "Preparation of peptide antigen for immunoassay".

The serum samples were from two different rabbits (rabbit 1 and rabbit2) and from one human (human 1) and from a pool of human sera fromdifferent individuals (human 2). The sera were used at a 1/500 dilution.The conjugate used was an alkaline phosphatase goat anti-rabbit Igconjugate (Sigma) diluted 1/500 or an alkaline phosphatase swineanti-human IgG conjugate (Orion Diagnostica) diluted 1/100.

The plates were read at times indicated below in an automatedELISA-reader (Titertek, Multiscan).

The results were as follows:

    ______________________________________                                                             Plates read                                                                              Absorbance at                                 Serum   Conjugate    after      405 nm                                        ______________________________________                                        Rabbit 1                                                                              goat anti-rabbit                                                                           3 min      1.21                                          Rabbit 2                                                                              goat anti-rabbit                                                                           3 min      1.05                                          Human 1 swine anti-human                                                                           20 min     1.11                                          Human 2 swine anti-human                                                                           20 min     1.26                                          none    goat anti-rabbit                                                                           5 min      1.23                                          none    swine anti-human                                                                           13 min     0.84                                          ______________________________________                                    

Since the above given results in all instances showed positive reactionsthese data support that the synthesized peptide reacts with both rabbitand human sera--in this latter instance both with serum from oneindividual (human 1) as well as with a pool of human sera (human2)--and/or in these instances used alkaline phosphatase goat anti-rabbit Ig conjugate and alkaline phosphatase swine anti-human IgGconjugate, respectively.

That the synthesized peptide reacted with and bound to the respectiveconjugate was further shown by the ability of the conjugates alone tobind the peptide. That this binding not was caused by interaction of thepeptide with alkaline phosphatase in these conjugates was proven in asimilar experiment where enzyme alone was used and no binding wasobserved.

SPECIFIC BINDING OF THE SYNTHESIZED PEPTIDE TO HUMAN IgG

The ability of the synthesized peptide to specifically bind to human IgGwas further shown in the following experiment.

The synthesized peptide (0.5 mg) was applied onto a Sepharose.sup.® 4 B(Pharmacia, Uppsala, Sweden) column. The CNBr-activated Sepharose.sup.®4 B gel (10 ml) previously had been reacted with an excess (800 mg) ofhuman IgG over night at room temperature to immobilize the IgG throughcovalent linkage. 150 mg of the human IgG was found to be covalentlyattached to the gel matrix.

Approximately 85% of the synthesized peptide was retained, i.e. bound tothis column at pH 7.2 in PBS at 4° C. as well as at 25° C. The fractionof the synthesized peptide that did not bind to the column most likelycomprises contaminants, i.e. peptides of other sequences, which arewell-known to occure when peptides are synthesized according toMerrifield.

In a control experiment only utilizing Sepharose.sup.® 4 B gel (10 ml)in the column the CNBr activated sites were blocked with ethanolamine (2ml, 1 M ethanolamine +10 ml PBS) at room temperature over night.

Then the synthesized peptide (0.5 mg) was applied onto the column andthe column was irrigated with PBS. All of the synthesized peptide wasrecovered from the eluate.

Hence the peptide did not bind to Sepharose.sup.® 4 B as such but to thehuman IgG bound to the Sepharose.sup.® 4 B gel.

By using such a column (i.e. Human IgG Sepharose.sup.® 4 B) it ispossible to fractionate the active peptide(s) from the inactivepeptide(s).

The active peptide fraction could be eluted from the column by additionof 1% (v/v) aqueous acetic acid. The thus eluted peptide(s) could afterrestoring to pH 7.2 again be shown to bind to the same column.

Taken together these experiments further support that the synthesizedpeptide indeed bound to human IgG in the above desribed ELISAexperiments.

ASSAY FOR LYMPHOCYTE OR THYMOCYTE PROLIFERATING ACTIVITY: LAF ASSAY

In this experiment the synthesized peptide according to the invention isshown to exhibit a lymphocyte or thymocyte proliferating activity whichis comparable to that exhibited by a well-known mitogen, i.e.phytohaemagglutinin.

Substances and Equipment Used in the Assays

    ______________________________________                                        Medium:                                                                       ______________________________________                                        90 ml    RPMI Medium, Flow Laboratories                                       10 ml    Fetal calf serum, Sigma, USA                                         2 ml     Aqueous L-Glutamine solution 14.6 g/l,                                        Sigma, USA                                                           1 ml     Sodium pyruvate (100 mM), Gibco Limited,                                      UK                                                                   1 ml     Penicillin-Streptomycin (10000 units/ml),                                     Gibco Limited, UK                                                    ______________________________________                                    

Mitogen: Phytohaemagglutinin, PHA HA16 2 mg/5 ml H₂ O, WellcomeDiagnostics

(Methyl, 1',2'-³ H)-Thymidine: 4.44 Bq/nmol, 120 Ci/mmol, AmershamInternational plc, UK

Gas: air including 4% CO₂, Grundgas B-stand. OTC-20, AGA, Sweden

Centrifuge: IEC clinical centrifuge, 603 B

Climate chamber: WEDCO Incorporated, EZ-17MM

Incubation plates: NUNCLON Delta, InterMed, Denmark

Cell harvester: Dynatech, Automash 2000

Scintillation counter: Beckman LS-100C liquid scintillation system

Scintillation liquid: OptiPhase MP, LKB produkter AB, Sweden

Description of the Procedure

All the procedures were performed under sterile conditions.

The spleen was removed from a NMRI mouse and was placed in 5 ml ofmedium at room temperature. Then the spleen was homogenized through a100 mesh wire net. The homogenate was allowed to settle 2 min and thesuspension was decanted from the sediment, whereupon the cells werecentrifuged 8 min at 1220 rpm. The supernatant was discarded and thecells were washed once again by suspending in 5 ml medium andcentrifugation 8 min at 1220 rpm. The supernatant was discarded and thecells were suspended in 5 ml of medium. The cells were diluted 1/100 andcounted in a Burker chamber, and were then diluted to a finalconcentration of 5×10⁶ cells/ml.

LAF-Assay

The outer rows of wells in the incubation plates were not used forsamples but were filled with 200 μl medium in order to create a stabiletemperature zone. "Negative" controls were placed in 12 wells/plate andconsisted of 100 μl medium and 100 μl cell suspension. 100 μl sampleswere placed in new wells and a 1:2 serial dilution was made in theremaining wells of the plate. At least triplicates of the samples weremade. As a reference (control) the mitogen phytohaemagglutinin (PHA) wasused. Into separate wells were added in triplicate 2.5 μg, 5 μg and 7 μgof PHA in medium as well as 1 μg and 10 μg of the synthesized peptideaccording to the invention in medium.

The plate was placed in a desiccator having distilled water at thebottom, whereupon the air in the desiccator was replaced by the abovementioned gas in 2 min. The lid was placed onto the desiccator,whereupon the desiccator was placed in the climate chamber and incubatedat 37° C., 37% humidity for 48 hours. After the incubation 25 μl of ³H-thymidine was added to each well (0.010 μCi/well) and the plate wasfurther incubated 16 hours in the climate chamber.

Then the wells were quantitatively harvested by means of the cellharvester and the test samples were collected on filter paper. To ensurecomplete harvest of the cells the wells were washed three times with0.9% (w/v) aqueous sodium chloride solution. The thus harvested cells onfilter paper were stamped out and placed into scintillation vials. 6 mlscintillation liquid was added to each vial. The vials were agitated andwere allowed to stand for 15 min prior to measurement of theradioactivity in the scintillation counter for 5 min/vial.

    ______________________________________                                        Results:                                                                      Additions    cpm (range of triplicates)                                       ______________________________________                                        No mitogen   600-870                                                          PHA 2.5 μg                                                                              6700-8100                                                        PHA 5 μg   9800-10400                                                      PHA 7 μg  7000-8100                                                        The peptide                                                                   of invention:                                                                  1 μg     3000-4200                                                        10 μg     7000-8500                                                        ______________________________________                                    

The above results clearly show that the synthesized peptide according tothe invention exhibits a thymocyte proliferating activity which issimilar to that exhibited by the well-known mitogen PHA.

Immunization with the Synthetic Peptide

Enzyme-linked immunosorbent assay (ELISA) for measuring the reactionbetween native toxin and antibodies specifically induced by immunizationof mice with the synthetic peptide according to the invention.

The peptide used as antigen for immunization of mice was first treatedas described under "Preparation of peptide antigen for immunoassay".

Groups of mice (NMRI) were immunized subcutaneously with three doses onemonth apart, 3×0.5 ml, of the peptide antigen using aluminium hydroxideas carrier. Serum samples were collected two months after the thirddose. Microplates for ELISA were coated with purified pertussis toxin ata concentration of 1 μg/ml. Gelatin (1%) was used for blockingunspecific binding instead of BSA (c.f. "GENERAL DESCRIPTION OF ELISA").The endpoint titre of the sera was determined by ten two-fold dilutionsstarting from a 1/20 dilution and using an absorbance value of 0.1 ascut off. The conjugate used was an goat-anti mouse IgG conjugate (Sigma)diluted 1/500.

The plates were read after 30 minutes in an automated ELISA reader(Titertek, Multiscan).

The results from the assay were as follows:

    ______________________________________                                                No   Responders                                                                              Mean Titre Range                                       ______________________________________                                        The synthetic                                                                           17     15        2334     320-40960                                 peptide                                                                       Control   12      0         67      20-160                                    ______________________________________                                    

It is evident from the above results that the synthetic peptideaccording to the invention is capable of inducing antibodies uponimmunization, which have a clearly higher absorbance with thepostvaccination samples than with the prevaccination samples. Thus thetested peptide functions as an antigen and induces antibodies againstpertussis toxin.

As a consequence it can be established that the toxin specifically bindsto antibodies induced by the synthetic peptide in postimmunization serumsamples from mice.

Furthermore the same mice were challenged with purified pertussis toxin(20 μg/mouse=5×LD₅₀) and the mean days of survival was investigated inrelation to the endpoint titre.

    ______________________________________                                        Number      Days of                                                           of Mice     survival (mean)                                                                           Endpoint titre                                        ______________________________________                                        17          3.765        80-40960                                             15          4.000        320-40960                                            14          3.929        640-40960                                            10          4.600       1280-40960                                             8          4.875       2560-40960                                             6          5.333       5120-40960                                             3          6.333       10240-40960                                           Control     3.333                                                             12                                                                            ______________________________________                                    

As is evident from the above Table that the mean survival time increaseswith higher endpoint titres. The mice having high titres of antibodiesare thus partially protected against the challenge.

I claim:
 1. A polypeptide of the formula

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represent either no amino acid or an amino acidwhich facilitates coupling to a carrier, and Y represents --OH or --NH₂.2. An artificial compound in free or carrier--associated form whichbinds to glyco-conjugates, characterized in that it is chosen from thegroup consisting of the polypeptide of the formula

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represent either no amino acid or an amino acidwhich facilitates coupling to a carrier, and Y represents --OH or --NH₂; and functional analogues and functional derivatives of saidpolypeptide which bind to glyco-conjugates by possessing essentially thesame binding conformation in aqueous solution as said polypeptide.
 3. Anartificial compound in free or carrier--associated form which binds toimmunoglobulins, characterized in that it is chosen from the groupconsisting of the polypeptide of the formula

    H--X.sup.1 --Gln--Thr--Arg--Ala--Asn--Pro--Asn--Pro--Tyr--Thr----Ser--Arg--Arg--Ser--Val--Ala--Ser--X.sup.2 --Y

in which X¹ and X² each represent either no amino acid or an amino acidwhich facilitates coupling to a carrier, and Y represents --OH or --NH₂; and functional analogues and functional derivatives of saidpolypeptide which bind to immunoglobulins by possessing essentially thesame binding conformation in aqueous solution of said polypeptide.